Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-03
2002-07-02
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S071100, C435S071200, C435S471000, C435S325000, C435S320100, C435S252300, C536S023500, C530S350000
Reexamination Certificate
active
06413731
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Neuroregulators comprise a diverse group of natural products that subserve or modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids and lipid metabolites, and other metabolic byproducts. Many of these neuroregulator substances interact with specific cell surface receptors which transduce signals from the outside to the inside of the cell. G-protein coupled receptors (GPCRs) represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are characterized by seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequelae such as stimulation of adenylyl cyclase.
Opsins represent one of the major families of GPCRs. These receptors are unique compared to other GPCRs In that light is a crucial co-factor for their activation under physiological conditions. A major subclass of the opsin family is that of visual opsins such as rhodopsin and cone opsins. The visual opsins, also known as visual photopigments, are located in the eye and are involved in transducing visual information from the eye to the brain. Our understanding of opsin function has been derived primarily from the study of visual photopigments.
Rhodopsin and cone opsins are localized in retinal rod and cone photoreceptors, respectively. These photopigments respond to different wavelengths of light and thus have very distinct absorption spectra associated with different absorption maxima (&lgr;
max
) Even though both receptor subtypes convey visual signals to the brain in response to illumination, they have evolved to perform very distinct functions related to vision. Cone opsins are primarily responsible for color vision, also known as photopic vision, in different species. In contrast, rhodopsin, believed to have evolved from cone opsin, is mainly involved in dim light vision, also known as scotopic vision. Rhodopsin, highly enriched in rod photoreceptor membranes, has been used extensively as a model receptor to understand activation mechanism and functioning of opsins.
Rhodopsin contains the seven membrane-spanning apoprotein opsin and a retinoid-based chromophore (See reviews Hargrave and McDowell, 1992; Yarfitz and Hurley, 1994). In the ground or inactive state (i.e. in the absence of light), the chromophore, usually 11-cis-retinal, is covalently attached to a highly conserved lysine residue in the middle of the seventh transmembrane segment via a protonated Schiff base. All vertebrate visual opsins contain a highly conserved glutamate residue in the transmembrane helix 3 which serves as a counterion for the protonated Schiff base. It has been postulated that 11-cis-retinal behaves as an inverse agonist and induces an inactive conformation of the apoprotein which, by itself, is partially active (Cohen et al., 1993; Surya et al., 1995). Upon absorbing a photon, 11-cis-retinal is isomerized to the agonist all-trans-retinal which introduces distortion in the opsin and initiates a cascade of conformational changes in the molecule. Rhodopsin is first converted to bathorhodopsin, followed by lumirhodopsin, metarhodopsin I and metarhodopsin II states in a sequential manner. Even though most of these transient conformational states are difficult to study biochemically, they can be easily distinguished on the basis of their spectroscopic properties since each state has a unique absorption maximum. Experimental evidence suggests that the formation of metarhodopsin II, a relatively stable state, involves deprotonation of the Schiff base and represents the active conformation of the apoprotein. In this state, the opsin activates the cognate G-prozein and initiates the intracellular signaling cascade which ultimately results in transfer of visual information to the brain. Upon hydrolysis of the Schiff base linkage, metarhodopsin II decays into free all-trans-retinal and opsin. All-trans-retinal is transported to the neighbouring retinal pigment epithelial cells where it is converted to 11-cis-retinal via enzymatic reactions. 11-cis-retinal is transported back to retinal photoreceptors where it recombines with the opsin apoprotein to regenerate the rhodopsin molecule.
Even though all visual opsins essentially use the same activation mechanism as rhodopsin, there are some noticeable differences between vertebrate and invertebrate visual opsins (Gartner and Towner, 1995; Yarfitz and Hurley, 1994, Terakita et al., 1998; Arnheiter, 1998). Activation of vertebrate visual pigment results primarily in stimulation of G
t
G-protein (also known as transducin) leading to an increase in cGMP phosphodiesterase activity. Initiation of this signaling cascade ultimately results in closure of cation channels and hyperpolarization of the cell. In contrast, opsin visual pigments in invertebrates such as squid and fruitfly activate G
q
G-protein and elevate intracellular IP
3
and Ca
2+
levels (Wood et al., 1989; Nobes et al., 1992; Yarfitz and Hurley, 1994). Another major difference between vertebrate and invertebrate visual opsins is the stability of the active conformation of the receotor. Formation of vertebrate metarhodopsin II, the active conformation of rhodopsin, is rapidly followed by hydrolysis of the Schiff base linkage and dissociation of metarhodopsin II into free all-trans-retinal and opsin apoprotein. It has been suggested that the glutamate counterion in the transmembrane helix 3 aids in the hydrolysis reaction (Gartner and Towner, 1995). In contrast, invertebrate metarhodopsin represents a thermally stable state where the chromophore remains attached to the apoprotein (Kiselev and Subramaniam, 1994). This allows rapid photoisomerization of all-trans-retinal back to 11-cis-retinal within the apoprotein and rapid regeneration of rhodopsin, thus eliminating the need for retinal regenerating tissue (Provencio et al., 1998). The thermally stable metastate of invertebrate photopigment may be formed due to the absence of the glutamate counterion in transmembrane helix 3 of invertebrate visual opsins (Gartner and Towner, 1995).
Most opsins use 11-cis-retinal derived from carotenoids as a chromophore; however, some opsins use 3-hydroxy, 4-hydroxy or 3,4-dehydro isomers of 11-cis-retinal as a chromophore to accommodate the abundant availability of the substituted carotenoids (Gartner and Towner, 1995). Different opsins respond to photons with different wavelengths, a phenomenon known as spectral tuning. Even though the use of a particular retinal derivative as a chromophore contributes to spectral specificity (Gartner and Towner, 1995), the major determinant of spectral tuning is the presence of unique amino acids surrounding the retinal-binding site (Kochendoerfer et al., 1999). For example, substitution of a highly conserved glycine in transmembrane helix 3 of rhodopsin with amino acids of increasing size results in progressive shift of &lgr;
max
towards the blue wavelength (Han et al., 1996). Similarly, replacement of conserved non-polar residues with hydroxyl amino acids changes the opsin from a green-absorbing molecule to a red-absorbing pigment (Chan et al., 1992).
Even though the visual opsins have been at the forefront of opsin research, scientists are now turning their attention to non-visual opsins (the opsins not involved in transducing visual information) because of their potential involvement in physiological processes such as circadian rhythm and reproduction. The existence of non-visual photopigments in nonmammalian vertebrates was first suggested
Adham Nika
Borowsky Beth E.
Lakhlani Parul P.
Ogozalek Kristine L.
Cooper & Dunham LLP
Mertz Prema
Synaptic Pharmaceutical Corporation
White John P.
LandOfFree
Methods of screening for compounds which bind to a human... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods of screening for compounds which bind to a human..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of screening for compounds which bind to a human... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2907157